HPLC-DAD-MS Characterization, Antioxidant Activity, α-amylase Inhibition, Molecular Docking, and ADMET of Flavonoids from Fenugreek Seeds

Fenugreek (Trigonella foenum-graecum) has a great beneficial health effect; it has been used in traditional medicine by many cultures. Likewise, the α-amylase inhibitors are potential compounds in the development of drugs for the treatment of diabetes. The beneficial health effects of fenugreek lead us to explore the chemical composition of the seeds and their antioxidant and α-amylase inhibition activities. The flavonoid extraction from fenugreek seeds was achieved with methanol through a Soxhlet apparatus. Then, the flavonoid glycosides were characterized using HPLC-DAD-ESI-MS analysis. The antioxidant capacity of fenugreek seed was measured using DPPH, FRAP, ABTS, and CUPRAC assays. Finally, the α-amylase inhibition activity was carried out using in vitro and in silico methods. The methanolic extract was found to contain high amounts of total phenolics (154.68 ± 1.50 μg GAE/mg E), flavonoids (37.69 ± 0.73 μg QE/mg E). The highest radical-scavenging ability was recorded for the methanolic extract against DPPH (IC50 = 556.6 ± 9.87 μg/mL), ABTS (IC50 = 593.62 ± 9.35 μg/mL). The ME had the best reducing power according to the CUPRAC (A 0.5 = 451.90 ± 9.07 μg/mL). The results indicate that the methanolic extracts of fenugreek seed best α-amylase inhibition activities IC50 = 653.52 ± 3.24 μg/mL. Twenty-seven flavonoids were detected, and all studied flavonoids selected have good affinity and stabilize very well in the pocket of α-amylase. The interactions between the studied flavonoids with α-amylase were investigated. The flavonoids from fenugreek seed present a good inhibitory effect against α-amylase, which is beneficial for the prevention of diabetes and its complications.

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia as a result of a deficiency in insulin secretion or its action or both [17][18][19][20][21], which leads to disturbances in carbohydrate, fat, and protein metabolism [19].According to WHO, in 2014, there were 422 million adults living with diabetes worldwide.Diabetes mellitus (DM) accounted for 6.7 million deaths globally in 2021, with expenditures of USD 966 billion.Mortality is predicted to rise nearly 10-fold by 2030 [22] to a projected 783 million in 2045 [23].The rise of glucose levels in the blood is a result of carbohydrate hydrolysis, a process primarily catalyzed by the enzymes α-glucosidase and α-amylase [24].The process involves the hydrolysis of carbohydrates by the salivary α-amylase enzyme to disaccharides and oligosaccharides, which are further hydrolyzed by the α-glycosidase enzyme to monosaccharides like glucose, which are absorbed into the blood through the small intestine [25].The pancreatic α-amylase then hydrolyzes the remaining oligosaccharides to glucose and maltose [26][27][28].So, α-amylase is a key enzyme for starch digestion [29].Inhibition of α-amylase can help in reducing hyperglycemia [30].Pharmacologically, α-amylase inhibitors may be employed for the decrease in glucose absorption rate and, subsequently, reduce the postprandial rise in plasma glucose and the risk for long-term diabetes complications [31].Currently, α-amylase inhibitors such as acarbose are introduced to the market as a treatment for diabetes [26,32].The current anti-diabetes drugs can cause side effects such as current anti-diabetes drugs can cause side effects such as decreased appetite, diarrhea, stomach pain, and extreme tiredness, which reduce patient compliance and treatment effectiveness [33]; therefore, efforts to find natural inhibitory components that could replace these medications have gained more attention [34].Flavonoids are a group of compounds that can inhibit amylase enzyme activity [35].
Moreover, molecular docking has been frequently used in drug design, aiming to predict binding sites of a ligand with a target protein.The result of a ligand on the target protein could be predicted by comparing its binding site with an established drug (e.g., inhibitor), which has known action on that protein.Likewise, molecular docking has been applied to identify key active compounds that can bind to the corresponding targets at the known active site.To the best of our knowledge, the inhibitory effect of compounds from fenugreek against α-amylase was investigated for the first time using in silico methods in this study.
Oxidative stress is a common cause of many diseases, like diabetes, high blood pressure, preeclampsia, atherosclerosis, acute renal failure, Alzheimer's, and Parkinson's [36][37][38][39].It results from the loss of balance between oxidation and antioxidation processes, increasing free radicals in the body and leading to deficient cell operation [40,41], causing cell damage and dysfunction [42].Several studies demonstrate that antioxidants are important to maintain body health due to their capacity to neutralize free radicals, leading to a decrease in oxidative stress [43].The antioxidants can be found naturally in plants, microorganisms, and animals or chemically synthesized [43,44].The possible toxicological risks of synthetic antioxidants have been reconsidered, leading researchers to seek natural antioxidants that have more benefits and are less toxic [45][46][47].Natural antioxidants have gained significant attention due to their potential to improve antidiabetic therapy [48] and may reduce or prevent Type II diabetes through a number of processes, including (1) lowering mitochondrial oxidative stress, (2) decreasing the adverse effects of lipid peroxidation, and (3) contributing as important cofactors for antioxidant enzymes [49].
In this study, we aim to explore the chemical composition of crude extract of fenugreek seeds using HPLC-DAD-ESIMS analysis and investigate the antioxidant and antidiabetic effects with the aid of computational approaches followed by in vitro experiments.

TPC, TFC, and Antioxidant Activity
The pharmacological effects of fenugreek seeds may be a result of the several secondary metabolites presence, such as phenols and flavonoids.The extraction yield, total phenolics, and flavonoid content in the methanolic extract from fenugreek seeds obtained by Soxhlet are shown in Table 2. Secondary metabolites and composites extracted from plants have shown a beneficial impact on an individual's health, such as flavonoids and phenolic compounds.Thus, our study shed light on the fenugreek, where the yield of extraction for fenugreek seeds was 17.6%, using 10 g of seed powder (Table 2), which is consistent with [55] findings that reported a yield portion of 17.66%, using 10 g of fenugreek seeds, whereas [2] findings had shown much more yield, in which that latter is about 20.25% while the value ranged between 9.68% and 25.89%, using multiple organic solvents [56].Therefore, the yield of extraction can be influenced by many factors, such as genetic factors, environment, extraction manner, solvent type, and its polarity, as well as the ratio of the solvents with the plant matter [57][58][59].
Polyphenols are secondary metabolites naturally produced by plants [60] to be protected from biotic and abiotic stress [61].They have anti-inflammatory, antioxidant, and antimicrobial activities and anti-coronavirus properties [62].Table 2 illustrates the findings of the measured phenolic and flavonoid content in the methanol extract, in which the phenolic content was 154.68 ± 1.50 µg of GAE/mg of the extract.Meanwhile, the total fla-

TPC, TFC, and Antioxidant Activity
The pharmacological effects of fenugreek seeds may be a result of the several secondary metabolites presence, such as phenols and flavonoids.The extraction yield, total phenolics, and flavonoid content in the methanolic extract from fenugreek seeds obtained by Soxhlet are shown in Table 2. Secondary metabolites and composites extracted from plants have shown a beneficial impact on an individual's health, such as flavonoids and phenolic compounds.Thus, our study shed light on the fenugreek, where the yield of extraction for fenugreek seeds was 17.6%, using 10 g of seed powder (Table 2), which is consistent with [55] findings that reported a yield portion of 17.66%, using 10 g of fenugreek seeds, whereas [2] findings had shown much more yield, in which that latter is about 20.25% while the value ranged between 9.68% and 25.89%, using multiple organic solvents [56].Therefore, the yield of extraction can be influenced by many factors, such as genetic factors, environment, extraction manner, solvent type, and its polarity, as well as the ratio of the solvents with the plant matter [57][58][59].
Polyphenols are secondary metabolites naturally produced by plants [60] to be protected from biotic and abiotic stress [61].They have anti-inflammatory, antioxidant, and antimicrobial activities and anti-coronavirus properties [62].Table 2 illustrates the findings of the measured phenolic and flavonoid content in the methanol extract, in which the phenolic content was 154.68 ± 1.50 µg of GAE/mg of the extract.Meanwhile, the total flavonoid compounds were 37.69 ± 0.73 µg of QE/mg of the extract.Thus, the obtained findings were the best according to [63] results, in which they figured out that the total content of polyphenols and flavonoids of the ethanol extract was 9.7 mg of GAE/g of the extract, and 14.6 mg of QE/g of the extract respectively; hence the phenolic content is affected by the solvent and its polarity [64].Also, according to [65], methanol extract contains more phenolic and flavonoids than ethanol extract because methanol is more polar than ethanol.This divergence is also due to several factors, including type, environmental factors, and agriculture techniques [66].
Oxidation stress is associated with many diseases, such as diabetes, atherosclerosis, hypertension, respiratory diseases, arthritis, cataracts, cancer, cardiovascular disease, etc., that are caused by free radicals [67].The currently used antioxidants, such as BHA, BHT, PG, and TBHQ, are responsible for liver damage and cancer [68].
There is great interest in developing new antioxidants used in the pharmaceutical, cosmetic, and food industries.Also, botanical extracts with antioxidant capacity may work through various mechanisms, including oxygen root absorption, energy reduction, and free radical removal [69].Therefore, it is crucial to use several methods based on multiple chemical reactions to determine antioxidant activity [70].Accordingly, to examine methanolic extract antioxidant activity, four tests have been performed, namely, DPPH, ABTS, FRAP, and CUPRAC, and the findings are shown in Table 3.The DPPH and ABTS tests were used to measure the extract radical scavenging, where there was an equinox in the activity, which indicated an IC 50 equal to IC 50 = 556.6 ± 9.87 µg /mL and 593.62 ± 9.35 µg /mL, respectively.This result is stronger than that found by [71], in which IC 50 values varied between 80 and 149 mg/mL of the methanol extract of four fenugreek cultivars, But compared our finding with those of [72] with 172.6 ± 3.1 µg/mL in fenugreek seed oil was with DDPH test, our results still weak while, we had obtained better results with ABTS test, than those of [73], who stated that the IC 50 value was 962.5 µg /mL of the fenugreek seeds ethanol extract.While compared with [72], who found the antioxidant capacity of the oil by ABTS assay (IC 50 = 161.3± 2.21 µg/mL), our findings are weak.This is because antioxidant activity compounds dissolve in methanol better than ethanol because of methyl radical presence that is shorter than the ethyl radical present in ethanol, which leads to more solvation of the antioxidant molecules [74].
Besides, the antioxidant activity was also examined using FRAP and CUPRAC to assess reduction capability.On one hand, the value, using CUPRAC test, was A0.50 = 451.90± 9.07 µg /mL of methanol extract, while it was 108.33 mg TE/g extract of fenugreek seeds [75], the value that does not reach A0.50, using iron reduction test, in which the maximum focus of the test was 200 µg/mL, and the samples did not show up to 50% of the reduction capacity within this range; hence, its value "A0.50" was re-ported to be greater than 200 µg/mL.The antioxidant activity and the reduction capacity of fenugreek seeds methanol extract are owing to the presence of phenolic and flavonoid compounds.According to [76], fenugreek can have an antioxidant ability because there is flavone C-glycoside.
Similarly, the antioxidant impact is caused by the presence of flavonoid compounds, such as rutin, apigenin 7-glucoside, Quercetin [77], kaempferol [78], as well as the antioxidant activity per compound is all about many composition factors, namely: Phenols Number; Hydroxy groups or Methoxy groups; and other compositions [79].

Anti-α-amylase Activity
Diabetes, also known as diabetes mellitus, is a chronic disease caused by a group of metabolic disorders characterized by a high blood glucose level (hyperglycemia) [80] as a result of the lack of insulin production or its ineffectiveness.There are only two treatments for this disease, which are insulin injections or long-term intake of antidiabetic drugs [81].Therefore, α-amylase enzyme inhibitors are used to treat diabetes because they slow down the digestion of complex carbohydrates; consequently, glucose level gets low after eating [82].On the one hand, the α-amylase enzyme can be found at the level of both saliva and pancreas because it is responsible for dividing long-chain carbohydrates (starch) and converting them into maltose.It is the α-glucosidase pillar in the small intestine that facilitates its absorption, leading to hyperglycemia (high blood glucose) [83].On the other hand, In the case of diabetics, these enzyme inhibitors delay the complex carbohydrates division that leads to Diabetes hypoglycemia (a low level of glucose in the blood) after eating.[84]; thus, the α-amylase enzyme is the most important element that is needed in the digesting process of carbohydrates.Consequently, the inhibition of this enzyme leads to the inhibition of the digestion process of carbohydrates, resulting in diabetic hypoglycemia after eating [85].
Therefore, the current studies on anti-diabetic drugs have shown that botanical medicine has a great role in detecting high-potential plants to inhibit α-amylase enzymes.After all, an investigation on the anti-diabetes effect of fenugreek seed extract was carried out by assessing the methanol extract's potential to inhibit the amylase enzyme.This approach aims to prevent hypoglycemia after eating by interfering with the decomposition and absorption of carbohydrates in the intestine [86], and the anti-diabetes effect of T Foenum-graecum extract (Reported using IC 50 values) is illustrated in Table 2.
In addition, Methanol extract strongly inhibited the amylase enzyme compared to the positive control of acarbose, where for methanol extract, IC 50 was valued at 653.52 ± 3.24 µg/mL, while the acarbose recorded value was 3650.93 ± 10.70 µg/mL.
However, [87] findings were found to be in approximate agreement with our findings, in which the IC 50 value was 690 ± 0.01 µg/mL, whereas [88] did not notice any effect of methanol extract on the concentration of 2.5 mg/mL, in which IC 50 value used to inhibit α-amylase of aqueous, hydroalcohol, and ethanol extracts 6.476, 12.395, and 8.690 mg/mL, respectively [89].Also, the inhibition ratio in the concentration of 1 mg/mL was 37.7% [90], whereas in the [91] research study, the α-amylase inhibition ratio was 41.64%.In contrast with the study that was conducted by [92], which presented that there are relatively low molecular weight compounds in the water extract of fenugreek that inhibit α-amylase and Sucrase in rat intestines.
Finally, flavonoid-rich and phenols-rich extracts can contribute to diabetes management and complications [70,89].Also, phenols play a great role in inhibiting α-amylase and inhibiting the absorption of glucose in the intestine.[93][94][95][96][97]; consequently, it has been reported that flavonoids are the active biological principle of most hypoglycaemic and anti-diabetic medicinal plants [98,99], and because there are flavone C-glycosides compounds, digestive enzymes were inhibited [100,101].Also, fenugreek Methanol extract had presented that it has a high value of phenolic and flavonoid compounds that were 154.68 ± 1.50 µg of GAE/mg of the extract and 37.69 ± 0.73 µg of the QE/mg of the extract, respectively.Also, we noticed that there are flavanoid-C-glycoside compounds, such as vicenin, isoschaftoside, and isoorientin.Although the pathophysiology of diabetes is not fully understood, many studies have reported the role of free radicals in the pathogenesis of diabetes and its complications [102].We noted through our results that fenugreek seeds have strong antioxidant activity, as indicated by many studies [103].This is because the main bioactive compounds in fenugreek seeds are polyphenols [104], which may be beneficial for the prevention of diabetes and its complications [97].

ADMET and Drug-Likeness Evaluation
We evaluated the pharmacokinetic (absorption, distribution, metabolism, excretion, and toxicity) and drug-likeness properties of the flavonoids from fenugreek seeds to be accepted as a treatment for diabetes "see Table 4 below".Compounds C2 and C9 showed good drug-likeness characteristics; however, the rest of the flavonoids violated the Lipinski rule.All compounds were predicted to be non-substrates or non-inhibitors for CYP (CYP1A2, CYP2C9, CYP2D6, CYP2C19, and CYP3A4) except C5, C6, C8, and C10, which they were predicted to be P-gp Substrate.These properties are not particularly significant, as the target is found in the digestive tube.All studied compounds were also predicted to be non-carcinogens, while all of them showed potential hepatotoxicity and mutagenesis with a probability of 0.4 to 0.7, including the FDA-approved drug acarbose.

Molecular Docking
Molecular docking methods are commonly used in medicinal chemistry research for calculating the affinity of molecules toward protein targets and predicting the interactions within complexes [105].
Firstly, we run a validation process to verify the accuracy and precision of our docking program.We validated the behavior of the co-crystallized inhibitor: acarbose for A. oryzae α-amylase for human pancreatic α-amylase by a low RMSD value of 0.58 Å and 0.55 Å, respectively.Molecular docking of nine flavonoids from fenugreek seeds (C1-C9) and acarbose (C10) into the α-amylases active site was performed.The results are shown in Table 5, and their binding conformations are represented in Figures 2-4.The resultant data were regarded as valid since redocking the crystallized ligands produced good ligand superposition with RMSD less than 2 Å [106].
Based on the results presented in Table 5, the studied flavonoids were very close in binding scores and binding modes to the co-crystallized inhibitor (acarbose), especially schaftoside and quercetin 3-rhamnoside with binding scores of −8.7 and −8.6 Kcal/mol, respectively, towards human pancreatic α-amylase, indicating promising binding affinity with expected effective inhibition activity.As shown in Table 5, the present interaction showed the formation of hydrogen bonds between all nine studied flavonoids and residues in the active site of α-amylases.The following hydrogen bonds (five) were formed between schaftoside and the human pancreatic α-amylase residues: Thr163 (2.22 Å), Ile235 (2.42 Å), Trp59 (2.62 Å) and His305 (2.51; 2.79 Å) (Figure 3c).Schaftoside formed also six hydrogen bonds with human salivary α-amylase as follows: Ser163 (2.67 Å), Ile235 (2.05 Å), His305 (2.81 Å), Asp300 (2.40; 2.84 Å) and Glu233 (2.18 Å) (Figure 5c), and six hydro- According to these observations, we suggest that schaftoside and quercetin 3-rhamnoside are good inhibitors for α-amylase and could be promising molecules to develop new drugs against diabetes.It is worth mentioning that the other flavonoids have also presented a good binding score and binding mode similar to acarbose, where they formed a significant number of hydrogen bonds and hydrophobic interactions with amino acids of the active site.

Molecular Dynamics Simulation
The molecular dynamic simulation was performed to examine the structural stability of the protein complexes and estimate the different bonds formed between the protein target and small molecules during 100 ns MD simulation.MD simulation revealed that the stable structures were preserved, as shown in Figures 5 and 6.The human pancreatic α-amylase in complex with schaftoside was the most stable with RMSD less than 2 Å.
amylase.The binding mode and the formed interactions with the amino acids of the binding pocket of the best-selected flavonoids and acarbose are represented in Figures 2-4.According to these observations, we suggest that schaftoside and quercetin 3-rhamnoside are good inhibitors for α-amylase and could be promising molecules to develop new drugs against diabetes.It is worth mentioning that the other flavonoids have also presented a good binding score and binding mode similar to acarbose, where they formed a significant number of hydrogen bonds and hydrophobic interactions with amino acids of the active site.

Molecular Dynamics Simulation
The molecular dynamic simulation was performed to examine the structural stability of the protein complexes and estimate the different bonds formed between the protein target and small molecules during 100 ns MD simulation.MD simulation revealed that the stable structures were preserved, as shown in Figures 5 and 6.The human pancreatic α-amylase in complex with schaftoside was the most stable with RMSD less than 2 Å.  2.6.1.RMSD Schaftoside and quercetin 3-rhamnoside exhibited a stable binding to human pancreatic α-amylase, as confirmed by minor changes in RMSD of the ligand (up to 1.5 Å) and protein backbone (up to 2 Å and 2.5 Å for schaftoside and quercetin 3-rhamnoside respectively), while RMSD of acarbose showed a big deviation between 10 and 40 ns (Figure 5b).For A. oryzae α-amylase, RMSD of the backbone increased gradually in the first 60 ns, then remained stable around 2.8 Å till the end of the simulation in the three cases (schaftoside, quercetin 3-rhamnoside, and acarbose) (Figure 5d).The backbone RMSD plot suggests that schaftoside and quercetin 3-rhamnoside show the same affinity towards human pancreatic α-amylase and do not disturb the structural stability of the enzyme.

RMSF
RMSF analysis of human pancreatic α-amylase showed low fluctuation around 3 Å except for residues 152, 349, and 350, which showed a notable fluctuation reaching 6 Å, respectively (Figure 5c).RMSF analysis of A. oryzae α-amylase also showed a small fluctuation of less than 2 Å except for residues 156-162, which showed a notable fluctuation that reached 8 Å respectively (Figure 5f).It has been confirmed that the loops can often present conformational changes [107,108].

Radius of Gyration
The radius of gyration (Rg) measures the compactness of a protein and shows the effect of the presence of a ligand on the protein structure [109].The constant values of Rg show that the ligand does not affect the protein structure, while the big fluctuations indicate the instability of protein folding and change of protein structure [110,111].The Rg plots of all studied complexes of both A. oryzae α-amylase and human pancreatic α-amylase were stable throughout 100 ns MD simulation (Figure 6a,b).These results indicated that the structural stability did not deteriorate during the MD simulation, and the enzymes preserved their native structure and were not affected by the ligands during the simulation.

Hydrogen Bonds
The binding affinity of schaftoside, quercetin 3-rhamnoside, and acarbose towards both types of α-amylases was confirmed by checking the intermolecular hydrogen bonds, where the formation of hydrogen bonds in the complex leads to strong binding [112].
MD simulation revealed that schaftoside, quercetin 3-rhamnoside, and acarbose interacted strongly with human pancreatic α-amylase, where they formed a minimum of 3 hydrogen bonds with active site residues during 100 ns MD simulation (Figure 6b).Whereas A. oryzae α-amylase schaftoside, quercetin 3-rhamnoside, and acarbose also formed several hydrogen bonds around six hydrogen bonds at the beginning of the simulation and about 3 HBs at the end of the simulation (Figure 6d).However, the stability of the studied molecules over time can be explained by the hydrogen bonds formed in the complexes alongside the high number of hydrophobic interactions formed between the ligands and α-amylases.

Principal Component Analysis (PCA)
PCA analyses were performed on the MD trajectories of 2QV4 with the QU and AC ligands.The total motions of backbone atoms were dispersed over 4456 eigenvectors.The combined contributions of the eigenvectors with the top two eigenvalues were 44% and 52% for the 2QV4 molecule with the QU and AC, respectively.PC1 and PC2 were plotted together to visualize the essential sub-space of collective motions (Figure 7).The eigenvalues taken from the diagonalization of the covariance matrix of the fluctuation of backbone atoms.The overall flexibility of both QU-2QV4 complex and AC-2QV4 complex was seen by the trace of the covariance matrix offer diagonalizing obtained for QU-2QV4 is 18.7302 nm2 and for AC-2QV4 17.6181 nm2.It can be observed that the range of the PC1 values is a little larger for the 2QV4-QU complex compared to other AC-2QV4 complexes (Figure 7A), indicating that the 2QV4-QU is relatively more flexible with respect to the motion depicted by eigenvector 1 (Figure 8A).For QU-7TAA and AC-7TAA complexes, PCA analysis was performed, and combined contributions of the eigenvectors with the top two eigenvalues were 49% and 59%, respectively.The plots of PC1 against PC2 of QU-7TAA and AC-7TAA are presented in Figure 7B.The total flexibility for both QU-7TAA complex and AC-7TAA complex was seen by the trace of the covariance matrix offer diagonalizing were 18.4854 nm2 and for AC-2QV4 23.3184 nm2.
52% for the 2QV4 molecule with the QU and AC, respectively.PC1 and PC2 were plotted together to visualize the essential sub-space of collective motions (Figure 7).The eigenvalues taken from the diagonalization of the covariance matrix of the fluctuation of backbone atoms.The overall flexibility of both QU-2QV4 complex and AC-2QV4 complex was seen by the trace of the covariance matrix offer diagonalizing obtained for QU-2QV4 is 18.7302 nm2 and for AC-2QV4 17.6181 nm2.It can be observed that the range of the PC1 values is a little larger for the 2QV4-QU complex compared to other AC-2QV4 complexes (Figure 7A), indicating that the 2QV4-QU is relatively more flexible with respect to the motion depicted by eigenvector 1 (Figure 8A).For QU-7TAA and AC-7TAA complexes, PCA analysis was performed, and combined contributions of the eigenvectors with the top two eigenvalues were 49% and 59%, respectively.The plots of PC1 against PC2 of QU-7TAA and AC-7TAA are presented in Figure 7B.The total flexibility for both QU-7TAA complex and AC-7TAA complex was seen by the trace of the covariance matrix offer diagonalizing were 18.4854 nm2 and for AC-2QV4 23.3184 nm2.

Free-Energy Landscape
Information on FEL is used to determine the most energetically favorable structure [113].The minima of global energy states are shown in violet.Figure 9A shows the FEL of (A) 2QV4-QU complex and (B).As can be seen, the 2QV4-AC complex displayed a single dominant minimum energy basin observed.2QV4-QU (Figure 9B) contains four clear single points of the lowest Gibbs energy, but with the single lowest energy that is much broader than the basins of 2QV4-AC.When QU and AC bound to 7TAA, FEL showed three distinct minima regions for QU (Figure 9C), and FEL also showed three distinct minima regions for AC (Figure 9D).

Free-Energy Landscape
Information on FEL is used to determine the most energetically favorable structure [113].The minima of global energy states are shown in violet.Figure 9A shows the FEL of (A) 2QV4-QU complex and (B).As can be seen, the 2QV4-AC complex displayed a single dominant minimum energy basin observed.2QV4-QU (Figure 9B) contains four clear single points of the lowest Gibbs energy, but with the single lowest energy that is much broader than the basins of 2QV4-AC.When QU and AC bound to 7TAA, FEL showed three distinct minima regions for QU (Figure 9C), and FEL also showed three distinct minima regions for AC (Figure 9D).Gibbs free energy landscape calculated from PC1 and PC2 of (A) 2QV4-QU complex, (B) of 2QV4-AC complex, (C) 7TAA-QU complex, and (D)of 7TAA-AC complex.

Plant Material
The fenugreek (Trigonella foenum-graecum L.) seeds were harvested in the Bin El Ouiden region in the spring of 2012 (Skikda Province, northeastern Algeria), cleaned, and airdried.Ten g of seeds were ground into a fine powder using an electric grinder and then stored at 4 °C until analysis.

Plant Material
The fenugreek (Trigonella foenum-graecum L.) seeds were harvested in the Bin El Ouiden region in the spring of 2012 (Skikda Province, northeastern Algeria), cleaned, and air-dried.Ten g of seeds were ground into a fine powder using an electric grinder and then stored at 4 • C until analysis.

Plant Extract Preparation
The extraction of fenugreek seeds was performed using the Soxhlet apparatus; ten grams (10 g) of powdered fenugreek seeds were placed in a Soxhlet apparatus and extracted thoroughly in a Soxhlet apparatus in three steps.The plant material was first defatted with hexane, then chloroform, and finally methanol.The obtained methanol extract was filtered and concentrated to dryness using a rotary evaporator.Then, the extraction yield was evaluated according to [114]  The total phenolic content was determined using Folin-Ciocalteu reagent according to the method described by [115].As follows: 20 µL of the sample was mixed with 100 µL of Folin-Ciocalteu reagent (10%), and 75 µL of aqueous sodium carbonate solution (7.5%, w/v), then the mixture was incubated at room temperature and in the dark for 2 h.The absorbance was measured at 765 nm using a microplate reader of type PerkinElmer (Enspire).Gallic acid was used as a standard for plotting the calibration curve.The total phenolic content was expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight.

Determination of Flavonoid Content (FC)
The total flavonoid content was determined according to the [116] method.Quercetin was used as a standard to plot the calibration curve.50 µL of sample or standard was incubated with 130 µL of methanol, 10 µL of potassium acetate (9.8%, w/v), and 10 µL aluminum nitrate nonahydrate (Al (NO 3 ) 2 , 9H 2 O) (10%, w/v), in room temperature and dark for 40 min.After incubation, the absorbance was measured at 415 nm using a microplate reader of type PerkinElmer (Enspire).The flavonoid content was expressed as milligrams of quercetin equivalents (QE) per g dry weight.

Anti-α-amylase Assay
The inhibition assay of methanolic extracts of Fenugreek seeds was carried out using the iodine/potassium iodide (IKI) method [117] with some modifications, as follows: 25 µL of extracts at different concentrations were added to 50 µL of A. oryzae α-amylase enzyme (1U), and the mixture was preincubated for 10 min at 37 • C.Then, the reaction was initiated by adding 50 µL of starch (0.1%).After 10 min of incubation at 37 • C, the reaction was stopped by adding 25 µL of HCl (1M) and 100 µL of IKI, and then the absorbance was measured at 630 nm using a microplate reader of type PerkinElmer (Enspire).The inhibition of α-amylase was calculated using the following equation: where The FDA-approved drug acarbose was used as a reference.The inhibitory activity of the extracts and standards was expressed as IC 50 (the concentration of the tested samples that inhibits 50% of α-amylase).

DPPH Assay
The ability of radical scavenging was determined according to [118], using the radical DPPH• (2,2-diphenyl-1-picrylhydrazyl).160 µL of DPPH solution (0.15 mM) was added to 40 µL of the diluted extracts in methanol and incubated in the dark.After 30 min of incubation, the absorbances were measured at 517 nm by a microplate reader of type PerkinElmer (Enspire) against a blank.BHA, BHT, and α-tocopherol were used as reference antioxidants.DPPH inhibition percentage was calculated using the following equation: where A0 is the absorbance of the control (without the sample) and As is the absorbance of the tested samples.The antioxidant activity of the extracts and standards was expressed as IC 50 (the concentration of the tested samples that inhibits 50% of DPPH radicals).

ABTS+ Scavenging Activity
The ABTS•+ assay (2, 2 -azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) was carried out according to the [120] method with some modifications.The ABTS+ reagent was prepared as follows: 19.2 mg of ABTS (7 mM) in 5 mL distilled water was mixed with 3.3 mg of Potassium persulfate K 2 S 2 O 8 (2.45 mM) in 5 mL distilled water, and the mixture was incubated for 16 h in the dark before use.We have added 160 µL of ABTS+ reagent to 40 µL of extract solutions.After 10 min of incubation, the absorbance was read at 734 nm using a microplate reader of type PerkinElmer (Enspire).BHA and BHT were used as references.The total antioxidant activity of the extracts was calculated as follows: 3.4.4.Cupric Reducing Antioxidant Capacity (CUPRAC) Assay Cupric reducing antioxidant capacity was determined using the [121] method.40 µL of the extracts were mixed with 60 µL of ammonium acetate (NH 4 CH 3 CO 2 ), and 50 µL of copper (II) chloride dihydrate (CuCl 2 , 2H 2 O), then 50 µL of neocuproine was added.After 1 h of incubation, the absorbance was read at 450 nm using a microplate reader of type PerkinElmer (Enspire).BHA and BHT were used as references.

ADMET and Drug-Likeness Evaluation
ADME/Tox evaluation is an important process for selecting a good drug candidate [122].The drug-likeness and toxicity of any compound can be evaluated using webservers that calculate some properties of a molecule based on its structure.In this study, we used two web servers: The swissADME server (http://www.swissadme.ch/index.php[123] (accessed on 15 January 2023)) and the admetSAR 2.0 server http://lmmd.ecust.edu.cn/admetsar2)[124] (accessed on 15 January 2023).We obtained The SMILES codes of the studied compounds (Figure 10) from the PubChem Database [125].

ADMET and Drug-Likeness Evaluation
ADME/Tox evaluation is an important process for selecting a good drug candidate [122].The drug-likeness and toxicity of any compound can be evaluated using webservers that calculate some properties of a molecule based on its structure.In this study, we used two web servers: The swissADME server (http://www.swissadme.ch/index.php[123] (accessed on 15 January 2023)) and the admetSAR 2.0 server http://lmmd.ecust.edu.cn/ad-metsar2)[124] (accessed on 15 January 2023).We obtained The SMILES codes of the studied compounds (Figure 10) from the PubChem Database [125].

Molecular Docking
We have performed a specific molecular docking simulation to study the interactions between the compounds (Figure 10) from fenugreek seeds and α-amylase enzymes.The enzyme α-amylase is considered the main target for the development of anti-diabetic

Molecular Docking
We have performed a specific molecular docking simulation to study the interactions between the compounds (Figure 10) from fenugreek seeds and α-amylase enzymes.The enzyme α-amylase is considered the main target for the development of anti-diabetic drugs.We have prepared the input files using Autodock tools (ADT) (version 1.5.4)[126] as follows: We have downloaded the crystal structure of α-amylases (PDB IDs: 3DHP, 2QV4, and 7TAA) from Protein Data Bank (PDB) [127].The files were assembled, and then we removed the water molecules, co-crystallized solvent, ligands, and other heteroatoms.We have added polar hydrogens and partial charges, and the docking box was set based on the co-crystallized inhibitor.The size of the box and centers are presented in Table 6.Finally, we have run the molecular docking using Autodock Vina 1.1.2software.We have set 50 runs with one conformation for each compound.After the docking finish, we loaded the output files into the Discovery Studio visualizer (4.0).The most repeated conformation with less binding energy was considered the most stable and chosen for the analysis [128][129][130].

Molecular Dynamics Simulation
After the molecular docking, we checked the stability of the selected complex's protein-ligand (2QV4-acarbose, 2QV4-Schaftoside, 2QV4-QuercetinO-rhamnoside, 7TAAacarbose, 7TAA-Schaftoside, and 7TAA-QuercetinO-rhamnoside) by molecular dynamics simulation using Gromacs 2022 [131] according to the method described in [105].Firstly, we generated the ligands topology files using the CGenFF webserver (https: //cgenff.umaryland.edu)(accessed on 15 June 2022) [132] and target topology files using charmm-gui.Then, we put the complex in a rectangular box that contains water molecules (TPT3) and neutralized the system by adding Na+ ions.After the energy minimization, we heated the system to 300 K for 1000 picoseconds.The temperature and the pressure remained constant at around 300 K and 1 atm, respectively [133].Finally, we run the MD simulation for 100 ns.The recording was made for every 10 ps [134].After the simulation was finished, we analyzed the trajectory files using Gromacs build-in tools to study the dynamic conformational changes and the interaction in the complexes throughout time.We have used XMGRACE to plot the graphs [135].

Statistical Analysis
The results of the tests carried out are expressed as an average ± SD of analyses in three tests.The values of IC 50 (50% inhibition concentration) and A0.5 (the concentration indicating 0.50 absorbance) are calculated using the linear regression method from the two curves: [% inhibition = f (concentration)] for IC 50 and [Absorbance = f (concentration)] for A0.5.All the antioxidant and enzymatic tests were carried out at more than four concentration values.The one-way analysis of variance ANOVA was used to detect significant differences (p < 0.05).

Conclusions
Diabetes is a chronic metabolic disorder characterized by high blood glucose levels resulting from a deficiency in insulin secretion or its inability to function effectively.One promising approach to managing diabetes is through the use of natural plant-based remedies that can regulate blood sugar levels by inhibiting enzymes such as amylase, which breaks down carbohydrates into glucose.In this work, the methanolic fraction was extracted using the Soxhlet extraction technique, and the phytochemical profile, antioxidant, and α-amylase inhibitory obtained from the seeds of Trigonella foenum-graecum have been evaluated by using an HPLC-DAD-ESI-MS analysis, twenty-seven flavonoid compounds were identified, including twenty-two flavones and three flavonols.Higher levels of phenolic compounds in fenugreek showed stronger antioxidant activities and inhibitory potency of amylase enzyme.The major flavonoid compounds that were found in the methanolic extract of fenugreek seeds showed good amylase enzyme inhibitory activities.Molecular docking analysis revealed the underlying inhibition mechanisms of these compounds against amylase enzyme; the studied flavonoids were also very close in binding scores and binding patterns of acarbose towards human α-amylase, indicating a promising binding affinity with expected potent inhibition activity.The findings of this study would urge more research into high-degree isolation, structural re-determination, and in-depth bio-assaying of the most promising compounds.

Figure 6 .
Figure 6.The radius of gyration and the number of hydrogen bonds of the studied complexes.(a,c): Total Rg change over time, (b,d) Number of hydrogen bonds during the 100-ns MD.

Figure 7 .
Figure 7. (A) PCA plots show the projections of the displacement of the backbone of 2QV4 in complexed with QU (black) and of 2QV4 in complexed with ac (red), and (B) PCA plots show the

Figure 7 .
Figure 7. (A) PCA plots show the projections of the displacement of the backbone of 2QV4 in complexed with QU (black) and of 2QV4 in complexed with ac (red), and (B) PCA plots show the projections of the displacement of the backbone of 7TAA in complexed with QU (black) and of 7TAA in complexed with AC (red) 7TAA.Molecules 2023, 28, x FOR PEER REVIEW 15 of 26

Figure 8 .
Figure 8.The cumulative contribution of the 14 most relevant eigenvectors to the variance of the overall motion of (A) 2QV4 complexed with QU (black) and 2QV4 complexed with AC (red), (B) 7TAA in complexed with QU (black) and of 7TAA in complexed with AC (red) 7TAA.

Figure 8 .
Figure 8.The cumulative contribution of the 14 most relevant eigenvectors to the variance of the overall motion of (A) 2QV4 complexed with QU (black) and 2QV4 complexed with AC (red), (B) 7TAA in complexed with QU (black) and of 7TAA in complexed with AC (red) 7TAA.

Figure 10 .
Figure 10.Structure of flavonoids detected in methanolic extract of fenugreek seeds.

Figure 10 .
Figure 10.Structure of flavonoids detected in methanolic extract of fenugreek seeds.

Table 2 .
The extraction yield, total phenolic content, flavonoid content, and ani-α-amylase of the methanolic extract from fenugreek seeds.

Table 1 .
Chromatographic and spectral data of C-glycosylflavones identified by HPLC-DAD-ESI-MS in the fenugreek seeds.

Table 3 .
The antioxidant activities of the methanolic extract from fenugreek seeds.

Table 5 .
The molecular docking results: Repeating ratio of the chosen conformation, binding scores, and amino acid interactions of nine flavonoids from fenugreek seeds (C1-C9) and acarbose (C10) in the binding pocket of three types of α-amylases (A.oryzae, human pancreatic, and human Salivary).